With the advent of plant genetic engineering technology, the ecological implications of genetically modified crops are of great concern, particularly when there are no inherent barriers to the spread of transgenes through sexual reproduction. Specifically, concerns have arisen in cases when transgenes can spread from a transgenic plant to a weedy species through hybridization, or when the crop species itself exists in weedy forms. Bergelson et al. Nature 395: 25 (1998). One way to address such concerns is by genetically engineering sterility in a plant through complete ablation of reproductive structures.
Recently, there has been significant interest in using an ablation system for controlling reproductive development in plants. Reproductive control has been achieved in several plant species by genetic ablation, which entails linking a reproductive-preferred promoter with a cytotoxic gene to ablate reproductive cells. For example, barnase, an extracellular ribonuclease from Bacillus amyloliquifaciens has been employed for inducing male sterility. Paddon et al. J. Bacteriol. 171:1185-1187 (1989). European Patent No. 344,029 describes a system for producing a male sterile plant by transforming a plant with a DNA encoding barnase under the control of a tapetum-specific promoter. Transformation of tobacco and oilseed rape plants with such a promoter-gene construct prevented the plants from producing fertile pollen. Mariani et al., Nature 347: 737-741(1990). Flowers of transgenic Arabidopsis thaliana plants expressing a fusion construct of the APETALA3 (AP3) promoter and the diphtheria toxin A chain (DTA) gene lack petals and stamens, suggesting that transgene expression ablated petal and stamen cells. Transgenic Arabidopsis expressing the DTA gene under control of the LEAFY promoter produced no flowers. Tobacco plants transformed with a tobacco stigma-specific promoter driving the barnase gene lacked the stigmatic secretory zone and were female sterile.
Although genetic ablation has been effective, the promoters generally used for ablation are not well-suited for tissue-specific expression. As a consequence, leaky gene expression can significantly reduce and damage plant vegetative growth. Depending on the plant species, ablation can reduce vegetative growth by 80%. Strauss, S. H. and Meilan, R. TGERC Annual Report (1998). For genetic ablation to be commercially useful in the forestry industry, the amount of damage to vegetative tissues must be minimized to nominal levels.
While numerous patents and patent application publications disclose genetic ablation using a variety of promoters and cytotoxic genes, there is little disclosure addressing the effects of ablation on a plant's vegetative growth and development. The LFY promoter from Arabidopsis, which is expressed strongly in floral meristems and weakly in developing leaves, has been used for producing plants with ablated flowers. Nilsson et al., Plant J. 15:799-804 (1998). However, very few plants transformed with LFY had ablated flowers and uncompromised vegetative development. Therefore, it would be impractical to use a similar approach for reproductive ablation in a tree species, since it would take years to produce, grow, and test many transgenic trees to identify those few trees that have sterility and normal vegetative growth.
The genetic ablation of a reproductive organ requires a delicate balance between promoter activity and ablation gene toxicity. While the barnase gene is widely used for ablation in plants, barnase-induced toxicity frequently causes detrimental effects on plant growth and development. Thus, it may be desirable to reduce the toxicity of barnase, such that reproductive ablation occurs without deleterious and unrecoverable damages to a plant's vegetative growth.
Concurrent with the production of a mutant barnase having reduced toxicity, it may also be desirable to minimize leaky expression of a reproductive ablation construct in a plant's vegetative tissues. By minimizing leaky or ectopic expression of a reproductive ablation construct in a plant, expression of a mutant barnase gene in the vegetative tissues may be better tolerated by the plant due to attenuated ablation, which depends on promoter activity and RNase activity of a barnase mutant.
Accordingly, there exists a need for a reproductive ablation system having reduced barnase-induced toxicity and minimal leaky expression in a plant's vegetative tissues.